EP2046895A2 - Asymmetric fluoro-substituted polymethine dyes - Google Patents

Asymmetric fluoro-substituted polymethine dyes

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Publication number
EP2046895A2
EP2046895A2 EP07789086A EP07789086A EP2046895A2 EP 2046895 A2 EP2046895 A2 EP 2046895A2 EP 07789086 A EP07789086 A EP 07789086A EP 07789086 A EP07789086 A EP 07789086A EP 2046895 A2 EP2046895 A2 EP 2046895A2
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EP
European Patent Office
Prior art keywords
conjugate
btm
group
dye
fluorescence
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EP07789086A
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German (de)
English (en)
French (fr)
Inventor
Michael Edward Cooper
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GE Healthcare AS
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GE Healthcare AS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4842—Monitoring progression or stage of a disease

Definitions

  • the present invention relates to improved conjugates of biological molecules with an improved class of water-soluble, red to near infra-red (NIR) cyanine labelling dyes.
  • the dyes are asymmetric fluoro-substituted polymethines, and exhibit a high degree of photostability and reduced dye-dye quenching, as well as a high fluorescence quantum yield.
  • the conjugates are useful for in vivo optical imaging, as well as fluorescence detection methods.
  • pharmaceutical compositions containing the conjugates, kits for the preparation of such compositions, and methods of in vivo imaging using the conjugates are also disclosed.
  • Fluorescent dyes based on polymethine chromophores are characterised by strong absorption maxima which can extend over a wide wavelength range.
  • US Patent 6048982 (Waggoner) discloses luminescent cyanine dyes having the structure (1):
  • X and Y are independently selected from the group consisting of O, S and CH 3 -C-CH 3 ; m is an integer from 1-4 and at least one of the groups R 1 , R 2 , R 3 , R 4 and R 7 is a reactive group, reactive with amino, sulphydryl or hydroxy nucleophiles.
  • the number of methine groups linking the heterocyclic ring systems defines the absorption and emission maxima of the dyes.
  • the absorption maxima increase from 3 to 5 to 7 methine groups (CyTM3 to Cy5 to Cy7 respectively) by an increment of approximately lOOnm each.
  • groups R 1 to R 14 are equal or different and represent in each case H, Cl, Br, an aliphatic or mononuclear aromatic group, each having at most 12 carbon atoms which may contain as a substituted group in addition to carbon and hydrogen, up to 4 oxygen atoms and 0, 1 or 2 nitrogen atoms or a sulfur atom, or a sulfur and a nitrogen atom or represents an amino function, having a nitrogen atom to which there is bound, hydrogen or at least one substituent having up to 8 carbon atoms, the substituent selected from the group consisting of carbon, hydrogen and up to two sulfonic acid groups, and n is zero, 1 or 2. At least one of R 1 to R 14 may contain a reactive group.
  • Waggoner et al [Org.Lett, 6(6), 909-912(2004)] described a polyfluoro- thiadicarbocyanine dye (3) having good photostability in aqueous solvents.
  • the dye exhibited reduced aggregation, enhanced quantum yield and greater resistance to photobleaching when compared with a non-fluorinated analogue:
  • the dyes of the present invention possess one or more sulfonic acid groups attached to the 1- or 3 -position of the indolium ring system.
  • the present dyes exhibit increased photostability, and reduced dye-dye interactions.
  • the increased photostability and reduced dye-dye interaction resulting in enhanced brightness are particularly useful for in-vivo applications, as for example in endoscopic imaging, where the light intensity and the related photobleaching are high and the informative fluorescent signal being limited should be separated from the unfavourable tissue autofluorescence and background signal.
  • the improved chemical stability of the present dyes will confer improved resistance to various biochemical processes that occur within living organism (such as enzymatic degradation), that will potentially reduce the toxicity and/or imaging inefficacy due to the metabolism of the dye-[biological targeting moiety] conjugate.
  • the dyes are also useful for in vitro assays involving fluorescence detection where continual excitation is a requirement, for example in kinetic studies, or in microarray analyses where microarray slides may need to be reanalysed over a period of days.
  • Cy D is a cyanine dye of Formula II:
  • Q is a group containing 1 , 2 or 3 carbon-carbon double bonds which forms a conjugated system with B;
  • R 1 , R 6 and R 7 are selected independently from C 1- ⁇ alkyl or -(CH 2 ) Ic -SO 3 M 1 ;
  • R 2 , R 3 , R 4 and R 5 are selected independently from H, F, -SO 3 M 1 and
  • B is an aromatic chrornophore selected from benzo[b]pyrilium, quinolinium and acridinium chromophores; with the proviso that at least one of R 2 , R 3 , R 4 and R 5 is F or -(CF 2 ) m -F.
  • Alkyl is a straight or branched chain alkyl group containing from 1-4 carbon atoms, for example methyl, ethyl, «-propyl, zs ⁇ -propyl and ⁇ -butyl and ⁇ -butyl.
  • Aryl is an aromatic substituent containing one or two fused aromatic rings containing 6 to 10 carbon atoms, for example phenyl or naphthyl, the aryl being optionally and independently substituted by one or more substituents, for example halogen, C 1-4 alkyl or C 1-4 alkoxyl.
  • Alkoxyl is a C 1-4 alkoxy substituent for example methoxy, ethoxy, propoxy and n- butoxy.
  • Heteroaryl is a mono- or bicyclic 5- to 10- membered aromatic ring system containing at least one heteroatom which may be selected from N, O, and S and is optionally and independently substituted by one or more substituents, for example halogen, straight or branched C 1-4 alkyl or C 1-4 alkoxyl.
  • Alkyl is a C 1-4 alkyl group substituted by an aryl or heteroaryl group as hereinbefore defined.
  • Halogen and halo groups are selected from fluorine, chlorine, bromine and iodine.
  • biocompatible cation By the term “biocompatible cation” (B c ) is meant a positively charged counterion which forms a salt with an ionised, negatively charged group (in this case a sulfonate group), where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.
  • suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion.
  • Preferred biocompatible cations are sodium and potassium, most preferably sodium.
  • BTM biological targeting moiety
  • the BTM preferably comprises: 3-100 mer peptides or peptide analogues which may be linear peptides or cyclic peptides or combinations thereof; or enzyme substrates, enzyme antagonists or enzyme inhibitors; synthetic receptor-binding compounds; oligonucleotides, or oligo-DNA or oligo-RJNA fragments.
  • peptide is meant a compound comprising two or more amino acids, as defined below, linked by a peptide bond (ie. an amide bond linking the amine of one amino acid to the carboxyl of another).
  • peptide mimetic or “mimetic” refers to biologically active compounds that mimic the biological activity of a peptide or a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between amino acids).
  • peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.
  • peptide analogue refers to peptides comprising one of more amino acid analogues, as described below.
  • amino acid is meant an L- or D-amino acid, amino acid analogue (eg. naphthylalanine) or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Conventional 3-letter or single letter abbreviations for amino acids are used herein. Preferably the amino acids of the present invention are optically pure.
  • amino acid mimetic is meant synthetic analogues of naturally occurring amino acids which are isosteres, i.e. have been designed to mimic the steric and electronic structure of the natural compound.
  • isosteres are well known to those skilled in the art and include but are not limited to depsipeptides, retro-inverso peptides, thioamides, cycloalkanes or 1,5- disubstituted tetrazoles [see M. Goodman, Biopolymers, 24, 137, (1985)].
  • Suitable enzyme substrates, antagonists or inhibitors include glucose and glucose analogues such as fluorodeoxyglucose; fatty acids, or elastase, Angiotensin II or metalloproteinase inhibitors.
  • a preferred non-peptide Angiotensin II antagonist is Losartan.
  • Suitable synthetic receptor-binding compounds include estradiol, estrogen, progestin, progesterone and other steroid hormones; ligands for the dopamine D-I or D-2 receptor, or dopamine transporter such as tropanes; and ligands for the serotonin receptor.
  • the BTM may be an affinity tag which is capable of binding specifically and non-covalently with its complementary specific binding partner, thereby forming a specific binding pair.
  • specific binding pairs include, but are not restricted to: biotin/avidin, biotin/streptavidin, polyhistidine tag-metal ion complexes with nitrilotriacetic acid (e.g. Ni 2+ : NTA).
  • the complementary specific binding partner may be one component of a labelling complex for detection of a target component. It is to be understood that in the context of the present invention, any two atoms or molecules that possess a specific binding affinity one for the other, may be employed.
  • affinity tags are selected from biotin, iminobiotin and desthiobiotin.
  • the cyanine dye (Cy 0 ) of Formula II is a fluorescent dye or chromophore which is capable of detection either directly or indirectly in an optical imaging procedure using light of green to near-infrared wavelength (500-1200 nm, preferably 600-1000 ran).
  • the Cy D has fluorescent properties.
  • Q is the group:
  • NIR dyes are those in which n is selected to be 2 or 3, most preferably 2.
  • j is preferably 1, ie. a linking group (L) is present.
  • linker group -(A) m - of Formula I one of the roles of the linker group -(A) m - of Formula I is to distance the Cy 0 from the active site of the BTM. This is particularly important because the Cy D is relatively bulky, so undesirable steric interactions could otherwise occur. This can be achieved by a combination of flexibility (eg. simple alkyl chains), so that the Cy D has the freedom to position itself away from the active site and/or rigidity such as a cycloalkyl or aryl spacer which orientate the Cy 0 away from the active site.
  • the nature of the linker group can also be used to modify the biodistribution of an in vivo imaging agent. Thus, eg.
  • the linker group may function to modify the pharmacokinetics and blood clearance rates of the imaging agent in vivo.
  • Such "biomodifier" linker groups may accelerate the clearance of the imaging agent from background tissue, such as muscle or liver, and/or from the blood, thus giving a better diagnostic image due to less background interference.
  • a biomodifier linker group may also be used to favour a particular route of excretion, eg. via the kidneys as opposed to via the liver.
  • sugar a mono-, di- or tri- saccharide.
  • Suitable sugars include: glucose, galactose, maltose, mannose, and lactose.
  • the sugar may be functionalised to permit facile coupling to amino acids.
  • a glucosamine derivative of an amino acid can be conjugated to other amino acids via peptide bonds.
  • the glucosamine derivative of asparagine (commercially available from NovaBiochem) is one example of this:
  • Formula I denotes that the -(L)J[Cy 0 ] moiety can be attached at any suitable position of the BTM. Suitable such positions for the -(L) J -[Cy 0 ] moiety are chosen to be at positions away from that part of the BTM which is responsible for binding to the active site in vivo.
  • B is of Formula Ha:
  • Y is selected from O and 1ST— R , where R is selected from H, C 1- ⁇ alkyl and
  • R a , R b , R c , R d , R e , R f and R g are selected independently from Q, H, C 1-4 alkyl, C 6-I0 aryl, heteroaryl, aralkyl, Hal, sulphydryl, amino, C 1-4 alkyl-substituted amino, quaternary ammonium, -SO 3 M 1 , -OR 9 and -COOR 9 , where R 9 is selected from H and C i- 4 alkyl; Z represents an optional fused phenyl ring, such that R f and R s are attached to the
  • R 8 is preferably -(CH 2 ) ⁇ SO 3 M 1 .
  • B is a benzo[b]pyrilium chromophore, such that Y is O + , Z is absent, and R e is the preferred Q group described above, said dye having the Formula (III):
  • R b is preferably an amino group of formula -NR 10 R 11 , where R 10 and R 11 are independently H or C 1-4 alkyl, or wherein R 10 in combination with R a or R 11 in combination with R c , or both, form additional saturated or unsaturated 6- membered rings.
  • R g of Formula III is C 1 -C 4 alkyl, for example methyl, ethyl, w-propyl, zs ⁇ -propyl, n-butyl and t-butyl.
  • a second preferred embodiment of Formula Ha is where B comprises a quinolinium chromophore such that Y is N + -R 8 , Z is absent, together with the preferred Q group, said dye having a structure chosen from Formula (IVa) or (IVb) or (IVc):
  • a third preferred embodiment of Formula Ha is where B is an acridinium chromophore such that Y is N + -R 8 , Z is present, together with the preferred Q group as defined above, said dye having the Formula (V):
  • the compounds of the present invention comprise at least one, preferably two or more fluorine atoms substituted directly or indirectly onto the dye chromophore.
  • compounds of Formulae (II), (III), (IVa), (IVb), (IVc) and (V) may be substituted in the indolium ring system by a fluorine atom.
  • R , R , R 4 and R 5 groups preferably at least one, more preferably at least two, and most preferably at least three are chosen to be F. Any remaining groups R 2 , R 3 , R 4 and R 5 are preferably H. In a particularly preferred embodiment, each of R 2 , R 3 , R 4 and R 5 is F. Fluoro substitution of the dyes of the present invention has been found to improve dye photostability.
  • the compounds of Formulae (II), (III), (IVa), (IVb), (TVc) and (V) may include a C 1-4 perfluoroalkyl substituent of formula -(CF 2 VF, where m is an integer of value 1 to 4, in the indolium ring system at one, preferably not more than two of the R 2 , R 3 , R 4 and R 5 positions.
  • the remaining groups R 2 , R 3 , R 4 and R 5 are preferably selected from H or F.
  • the perfluoroalkyl substituent is trifluoromethyl, i.e. m is 1.
  • the Cy 0 dyes of the present invention may be substituted directly or indirectly with from two to four or more sulfonic acid groups, preferably between two and three sulfonic acid groups. These sulfonic acid groups are chosen from the -SO 3 M 1 and - (CH 2 XSO 3 M 1 substituents of Formula II.
  • the use of dyes substituted by fluorine and having three or more sulfonic acid groups for labelling BTM results in a labelled product in which there is reduced dye-dye aggregation and improved photostability, compared with dyes having no such substitutions.
  • the fluorescence emission intensity of a BTM so labelled with the preferred dyes of the present invention increases with the number of covalently attached dyes.
  • the [BTM] -(L) j - moiety is covalently attached at any suitable position of the Cy D , including B, Q or the R 1 to R 7 groups.
  • the [BTM]-(L) n - moiety either takes the place of an existing substituent (eg. the R 1 to R 7 groups), or is covalently attached to the existing substituent.
  • the [BTM]-(L)j- moiety is attached at one or more of positions R 1 , R 6 , R 7 , R 8 , R a , R b , R c , R d , R e , R f and R g of the Cy° of Formulae (II), (IH), (IVa), (IVb), (IVc) and (V).
  • the [BTM]-(L),- moiety is more preferably attached at one or more of the R 1 , R 6 or R 7 positions of the Cy D .
  • R 1 is -(L) j [BTM]; and one of R 6 and R 7 is
  • R 1 is -(CH 2 X-SO 3 M 1 ; and one of R 6 and R 7 is -(L) j [BTM] and the other is C 1-4 alkyl.
  • the BTM may be of synthetic or natural origin, but is preferably synthetic.
  • the term "synthetic" has its conventional meaning, ie. man-made as opposed to being isolated 10 from natural sources eg. from the mammalian body. Such compounds have the advantage that their manufacture and impurity profile can be fully controlled. Monoclonal antibodies and fragments thereof of natural origin are therefore outside the scope of the term 'synthetic' as used herein.
  • the BTM is preferably chosen from: a 3- 100 mer peptide, enzyme substrate, enzyme antagonist or enzyme inhibitor. BTM is most preferably a 3-100 mer peptide or peptide analogue. When the BTM is a peptide, it is preferably a 4-30 mer peptide, and most preferably a 5 to 28-mer peptide.
  • BTM is a peptide
  • preferred such peptides include:
  • ST refers to the heat-stable toxin produced by E.coli and other micro-organisms
  • - laminin fragments eg. YIGSR, PDSGR, IKVAV, LRE and 25 KCQAGTFALRGDPQG,
  • RGD Arg-Gly-Asp-containing peptides, which may eg. target angiogenesis [R.Pasqualini et al, Nat Biotechnol. 1997 Jun;15(6):542-6];
  • ⁇ 2 -antiplasmin precursor [M.Tone et al., J.Biochem, 102, 1033, (1987)]; beta-casein [L.Hansson et al, Gene, 139, 193, (1994)]; fibronectin [A.Gutman et al, FEBS Lett., 207, 145, (1996)]; thrombospondin- 1 precursor [V.Dixit et al, Proc. Natl. Acad.
  • angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe E. C. Jorgensen et al, J. Med. Chem., 1979, VoI 22, 9, 1038-1044
  • Angiotensin II Sar-Arg-Val-Tyr-Ile-His-Pro-Ile (R.K. Turker et al., Science, 1972, 177, 1203).
  • Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu;
  • M metabolism inhibiting group
  • PEG groups are described for the linker group (L), below.
  • Preferred such PEG groups are the biomodifiers of Formulae Biol or Bio2 (below).
  • Preferred such amino terminus M groups are acetyl, benzyloxycarbonyl or trifluoro acetyl, most preferably acetyl.
  • Suitable metabolism inhibiting groups for the peptide carboxyl terminus include: carboxamide, tert-bntyl ester, benzyl ester, cyclohexyl ester, amino alcohol or a polyethyleneglycol (PEG) building block.
  • a suitable M group for the carboxy terminal amino acid residue of the BTM peptide is where the terminal amine of the amino acid residue is N-alkylated with a C 1-4 alkyl group, preferably a methyl group.
  • Preferred such M groups are carboxamide or PEG, most preferred such groups are carboxamide.
  • the -(L)J[Cy 0 ] moiety may optionally be attached to the M group.
  • at least one peptide terminus has no M IG group, so that attachment of the -(L) j [Cy°] moiety at that position gives compounds of Formulae Va or Vb respectively.
  • Z 1 is attached to the N-terminus of the BTM peptide, and is H or M IG ;
  • Z 2 is attached to the C-terminus of the BTM peptide and is OH, OB C , or M IG , where B c is a biocompatible cation (as defined above).
  • B c is a biocompatible cation (as defined above).
  • Z 1 and Z 2 are preferably both independently M IG .
  • Preferred such M IG groups for Z 1 and Z 2 are as described above for the peptide termini.
  • the BTM peptide may optionally comprise at least one additional amino acid residue which possesses a side chain suitable for facile conjugation of the Cy D , and forms part of the (A) m residues of the linker group (L).
  • Suitable such amino acid residues include Asp or GIu residues for conjugation with amine- functionalised Cy D dyes, or a Lys residue for conjugation with a carboxy- or active ester- functionalised Cy D dye.
  • the additional amino acid residue(s) for conjugation of Cy 0 are suitably located away from the binding region of the BTM peptide, and are preferably located at either the C- or N- terminus.
  • the amino acid residue for conjugation is a Lys residue.
  • L When a synthetic linker group (L) is present, it preferably comprises terminal functional groups which facilitate conjugation to [BTM] and Cy 0 . Suitable such groups (Q a ) are described below.
  • L comprises a peptide chain of 1 to 10 amino acid residues, the amino acid residues are preferably chosen from glycine, lysine, arginine, aspartic acid, glutamic acid or serine.
  • L comprises a PEG moiety, it preferably comprises units derived from oligomerisation of the monodisperse PEG- like structures of Formulae Biol or Bio2:
  • p is an integer from 1 to 10.
  • a PEG-like structure based on a propionic acid derivative of Formula Bio2 can be used:
  • Bio2 where p is as defined for Formula Biol and q is an integer from 3 to 15. In Formula Bio2, p is preferably 1 or 2, and q is preferably 5 to 12.
  • preferred L groups when the linker group does not comprise PEG or a peptide chain, preferred L groups have a backbone chain of linked atoms which make up the -(A) m - moiety of 2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms being especially preferred.
  • a minimum linker group backbone chain of 2 atoms confers the advantage that the Cy D is well-separated so that any undesirable interaction is minimised.
  • the linking group L is preferably selected from:
  • M is selected from: -CHR'-, -NR'-, -O-, -S-, -Ar-, -C(O)-NR'- and -C(O)-O-;
  • R' is H or C 1-4 alkyl,
  • Ar is phenyl, optionally substituted with sulphonate,
  • p and r are integers of value 1 - 5.
  • Particularly preferred linking groups are those wherein M is selected from -CH 2 - and -CONH-.
  • BTM peptides which are not commercially available can be synthesised by solid phase peptide synthesis as described in P. Lloyd- Williams, F. Albericio and E. Girald; Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997.
  • the present invention provides a pharmaceutical composition which comprises the conjugate of the first aspect together with a biocompatible carrier, in a form suitable for mammalian administration.
  • the “biocompatible carrier” is a fluid, especially a liquid, in which the conjugate can be suspended or dissolved, such that the composition is physiologically tolerable, ie. can be administered to the mammalian body without toxicity or undue discomfort.
  • the biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyro gen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is isotonic); an aqueous solution of one or more tonicity-adjusting substances (eg. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (eg.
  • the biocompatible carrier is pyrogen-free water for injection or isotonic saline.
  • the conjugate and biocompatible carrier are each supplied in suitable vials or vessels which comprise a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula.
  • a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen or argon), whilst permitting addition and withdrawal of solutions by syringe or cannula.
  • a preferred such container is a septum-sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium).
  • the closure is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity.
  • Such containers have the additional advantage that the closure can withstand vacuum if desired (eg. to
  • Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm 3 volume) which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation.
  • Pre-filled syringes are designed to contain a single human dose, or "unit dose” and are therefore preferably a disposable or other syringe suitable for clinical use.
  • the pharmaceutical compositions of the present invention preferably have a dosage suitable for a single patient and are provided in a suitable syringe or container, as described above.
  • the pharmaceutical composition may optionally contain additional excipients such as an antimicrobial preservative, pH-adjusting agent, filler, stabiliser or osmolality adjusting agent.
  • an antimicrobial preservative is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds.
  • the antimicrobial preservative may also exhibit some bactericidal properties, depending on the dosage employed.
  • the main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such microorganism in the pharmaceutical composition.
  • the antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful microorganisms in one or more components of kits used to prepare said composition prior to administration.
  • Suitable antimicrobial preservative(s) include: the parabens, ie. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal.
  • Preferred antimicrobial preservative(s) are the parabens.
  • pH-adjusting agent means a compound or mixture of compounds useful to ensure that the pH of the composition is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [ie. tr ⁇ (hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof.
  • buffers such as tricine, phosphate or TRIS [ie. tr ⁇ (hydroxymethyl)aminomethane]
  • pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof.
  • the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.
  • filler is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation.
  • suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.
  • the pharmaceutical compositions of the second aspect maybe prepared under aseptic manufacture (ie. clean room) conditions to give the desired sterile, non- pyrogenic product. It is preferred that the key components, especially the associated reagents plus those parts of the apparatus which come into contact with the conjugate (eg. vials) are sterile.
  • the components and reagents can be sterilised by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma- irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred to sterilise some components in advance, so that the minimum number of manipulations needs to be carried out. As a precaution, however, it is preferred to include at least a sterile filtration step as the final step in the preparation of the pharmaceutical composition.
  • the pharmaceutical composition of the second aspect is preferably prepared from a kit, as described for the third aspect below.
  • the present invention provides a kit for the preparation of the pharmaceutical composition of the second aspect, said kit comprising the conjugate of the first aspect in sterile, solid form such that, upon reconstitution with a sterile supply of a biocompatible carrier as described in the second aspect, dissolution occurs to give the desired pharmaceutical composition.
  • the conjugate may be provided as a lyophilised powder in a suitable vial or container.
  • the lyophilisate is then designed to be reconstituted with the desired biocompatible carrier to the pharmaceutical composition in a sterile, apyrogenic form which is ready for mammalian administration.
  • a preferred sterile, solid form of the conjugate is a lyophilised solid.
  • the sterile, solid form is preferably supplied in a pharmaceutical grade container, as described for the pharmaceutical composition (above).
  • the formulation may optionally comprise a cryoprotectant chosen from a saccharide, preferably mannitol, maltose or tricine.
  • the present invention provides a functionalised dye useful in the preparation of the conjugate of the first aspect.
  • Said functionalised dye comprises the Cy 0 of Formula II wherein said Cy 0 further comprises a group Q a , wherein Q a is a reactive functional group suitable for conjugation to the BTM.
  • the Q a group is designed to react with a complementary functional group of the BTM, thus forming a covalent linkage between the Cy D and the BTM.
  • the complementary functional group of the BTM maybe an intrinsic part of the BTM, or may be introduced by derivatisation of the BTM with a bifunctional compound as is known in the art.
  • the BTM is used without derivatisation, so that the Q a group is preferably a reactive group.
  • Table 1 shows examples of reactive groups and their complementary counterparts: Table 1: Reactive Substituents and Complementary Groups Reactive Therewith.
  • activated ester or “active ester” is meant an ester derivative of the carboxylic acid which is designed to be a better leaving group, and hence permit more facile reaction with nucleophile, such as amines.
  • suitable active esters are: N-hydroxysuccinimide (NHS), pentafluorophenol, pentafluorothiophenol, /r ⁇ r ⁇ -nitrophenol and hydroxybenzotriazole.
  • Preferred active esters are N- hydroxysuccinimide or pentafluorophenol esters.
  • Examples of functional groups present in BTM such as proteins, peptides, nucleic acids carbohydrates and the like, include: hydroxy, amino, sulphydryl, carbonyl (including aldehyde and ketone) and thiophosphate.
  • Suitable Q a groups may be selected from: carboxyl; activated esters; isothiocyanate; maleimide; haloacetamide; hydrazide; vinylsulphone, dichlorotriazine and phosphoramidite.
  • Q a is: an activated ester of a carboxylic acid, an isothiocyanate, a maleimide or a haloacetamide.
  • Q a is preferably an activated ester, with especially preferred such esters including:
  • a preferred such substituent on the Cy° is the activated ester of an alkyl carboxylic acid, preferably a 5-carboxypentyl group.
  • Preferred such esters include:
  • Q a is preferably chosen from:
  • Peptide, protein or oligonucleotide BTM for use in the invention may be labelled at a terminal position, or alternatively at one or more internal positions.
  • fluorescent dye labelling reagents see "Non-Radioactive Labelling, a Practical Introduction", Garman, AJ. Academic Press, 1997; "Bioconjugation - Protein Coupling Techniques for the Biomedical Sciences", Aslam, M. and Dent, A., Macmillan Reference Ltd, (1998). Protocols are available to obtain site specific labelling in a synthesised peptide, for example, see Hermanson, G.T., “Bioconjugate Techniques", Academic Press (1996).
  • Cy D dyes of the present invention may be prepared by a process comprising reaction of: (a) a first compound having the formula (A):
  • groups R a to R g , Y and Z are as defined for the first aspect (above);
  • intermediate compounds (A), (Ha) and (C) may be reacted in a two step process.
  • an intermediate compound is first formed by reacting an indolium compound of formula (A) with a compound (C) suitable for forming the linkage, for example, a suitably substituted N,N'-diphenylformamidine, or malonaldehyde dianil, in the presence of acetic anhydride, to form a 2-anilinovinyl or 4-anilino-l,3-butadienyl quaternary salt.
  • the intermediate quaternary salt may be reacted with an aromatic heterocycle such as benzo[b]pyrilium, quinolinium, or an acridinium moiety having a suitably reactive methyl group.
  • an aromatic heterocycle such as benzo[b]pyrilium, quinolinium, or an acridinium moiety having a suitably reactive methyl group.
  • the reaction is performed in the presence of acetic anhydride and potassium acetate at ambient temperature.
  • Alternative intermediates for forming the polymethine linkage joining the heterocyclic ring systems are known and are described, for example in Hamer, F.M., "The Cyanine Dyes and Related Compounds", Interscience (1964).
  • the Cy D dyes of the present invention may optionally be modified to include charged or polar groups may be added to enhance the solubility of the compound in polar or nonpolar solvents.
  • carboxylic acid groups may be converted into esters and amide groups
  • N-alkylation of quinaldine, lepidine and acridine may be performed using alkyl halides such as methyl iodide, or with butane sultone, or with ⁇ -haloalkylcarboxylic acids.
  • CyTM is a trademark of GE Healthcare UK Limited.
  • the present invention provides a method of preparation of the conjugate of the first aspect, which comprises:
  • Covalent labelling of proteins is typically performed in an aqueous buffered medium, suitably bicarbonate at pH 9.0, at ambient temperature for a period of typically 1 hour.
  • the reaction is normally carried out in the dark.
  • the labelled protein can be separated from any unreacted dye by size exclusion chromatography, for example using SephadexTM as the stationary phase and phosphate buffer, pH 7.0 as the eluant.
  • size exclusion chromatography for example using SephadexTM as the stationary phase and phosphate buffer, pH 7.0 as the eluant.
  • the ratio of the amount or concentration of dye to BTM should be adjusted accordingly.
  • the present invention also relates to two-step labelling processes in which, in a first step, the Q a -functionalised Cy° of the fourth aspect binds to, and thereby labels a primary component, such as an antibody, protein, DNA probe, etc.
  • a primary component such as an antibody, protein, DNA probe, etc.
  • the fluorescently labelled primary component is then used as a probe for detection of a secondary component, such as an antigen for which the antibody is specific.
  • the method of preparation of the conjugate comprises either:
  • Y 1 is a carboxylic acid, activated ester, isothiocyanate or thiocyanate group; Y 2 is an amine group; Y is a maleimide group.
  • Y 2 is preferably a primary or secondary amine group, most preferably a primary amine group.
  • the thiol group of the BTM is preferably from a cysteine residue.
  • the BTM may optionally have other functional groups which could potentially react with the Cy° derivative, protected with suitable protecting groups, so that chemical reaction occurs selectively at the desired site only.
  • protecting group is meant a group which inhibits or suppresses undesirable chemical reactions, but which is designed to be sufficiently reactive that it may be cleaved from the functional group in question under mild enough conditions that do not modify the rest of the molecule. After deprotection the desired product is obtained.
  • Amine protecting groups are well known to those skilled in the art and are suitably chosen from: Boc (where Boc is tert-butyloxycarbonyl), Fmoc (where Fmoc is fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde [i.e. l-(4,4- dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e. 3-nitro-2-pyridine sulfenyl).
  • Suitable thiol protecting groups are Trt (Trityl), Acm (acetamidomethyl), t-Bu (tert- butyl), tert-Butylthio, methoxybenzyl, methylbenzyl or Npys (3-nitro-2 -pyridine sulfenyl).
  • the use of further protecting groups are described in 'Protective Groups in Organic Synthesis', Theodora W. Greene and Peter G. M. Wuts, (John Wiley & Sons, 1991).
  • Preferred amine protecting groups are Boc and Fmoc, most preferably Boc.
  • Preferred thiol protecting groups are Trt and Acm.
  • Cy D dyes of the invention are shown in Tables 2, 3 and 4:
  • the present invention provides a method of in vivo optical imaging of the mammalian body which comprises use of either the conjugate of the first aspect or the pharmaceutical composition of the second aspect to obtain images of sites of BTM localisation in vivo.
  • optical imaging is meant any method that forms an image for detection, staging or diagnosis of disease, follow up of disease development or for follow up of disease treatment based on interaction with light in the green to near- infrared region (wavelength 500-1200 nm).
  • Optical imaging further includes all methods from direct visualization without use of any device and involving use of devices such as various scopes, catheters and optical imaging equipment, eg. computer-assisted hardware for tomographic presentations.
  • the modalities and measurement techniques include, but are not limited to: luminescence imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; transmittance imaging; time resolved transmittance imaging; confocal imaging; nonlinear microscopy; photoacoustic imaging; acousto-optical imaging; spectroscopy; reflectance spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and fluorescence mediated diffuse optical tomography (continuous wave, time domain and frequency domain systems), and measurement of light scattering, absorption, polarization, luminescence, fluorescence lifetime, quantum yield, and quenching.
  • the green to near-infrared region light is preferably of wavelength 600-1000 nm.
  • the optical imaging method is preferably fluorescence endoscopy.
  • the mammalian body of the sixth aspect is preferably the human body.
  • Preferred embodiments of the conjugate are as described for the first aspect (above).
  • the Cy° dye employed is fluorescent, photostable, having a Stokes shift of greater than 20 nm, with a high quantum yield, and also is water soluble.
  • the conjugate or pharmaceutical composition has preferably been previously administered to said mammalian body.
  • previously administered is meant that the step involving the clinician, wherein the agent is given to the patient eg. as an intravenous injection, has already been carried out prior to imaging.
  • This embodiment includes the use of the conjugate of the first embodiment for the manufacture of a diagnostic agent for the diagnostic imaging in vivo of disease states of the mammalian body where the BTM is implicated.
  • a preferred optical imaging method of the sixth aspect is Fluorescence Reflectance Imaging (FRI).
  • FRI Fluorescence Reflectance Imaging
  • the agent of the present invention is administered to a subject to be diagnosed, and subsequently a tissue surface of the subject is illuminated with an excitation light - usually continuous wave (CW) excitation.
  • the light excites the dye molecule.
  • Fluorescence from the Cy D which is generated by the excitation light, is detected using a fluorescence detector.
  • the returning light is preferably filtered to separate out the fluorescence component (solely or partially).
  • An image is formed from the fluorescent light.
  • Usually minimal processing is performed (no processor to compute optical parameters such as lifetime, quantum yield etc.) and the image maps the fluorescence intensity.
  • the imaging agent is designed to concentrate in the disease area, producing higher fluorescence intensity. Thus the disease area produces positive contrast in a fluorescence intensity image.
  • the image is preferably obtained using a CCD camera or chip, such that real-time imaging is possible.
  • the wavelength for excitation varies depending on the particular Cy 0 dye used.
  • the apparatus for generating the excitation light may be a conventional excitation light source such as: a laser (e.g., ion laser, dye laser or semiconductor laser); halogen light source or xenon light source.
  • Various optical filters may optionally be used to obtain the optimal excitation wavelength.
  • a preferred FRI method comprises the steps as follows:
  • Cy 0 is detected using a fluorescence detector
  • the light detected by the fluorescence detector is optionally filtered to separate out the fluorescence component;
  • an image of said tissue surface of interest is formed from the fluorescent light of steps (ii) or (iii).
  • step (i) the excitation light is preferably continuous wave (CW) in nature.
  • step (CW) continuous wave
  • the light detected is preferably filtered.
  • An especially preferred FRI method is fluorescence endoscopy.
  • An alternative imaging method of the sixth aspect uses FDPM (frequency-domain photon migration). This has advantages over continuous-wave (CW) methods where greater depth of detection of the EVi within tissue is important [Sevick-Muraca et al, Curr.Opin.Chern.Biol., 6, 642-650 (2002)]. For such frequency/time domain imaging, it is advantageous if the Cy 0 has fluorescent properties which can be modulated depending on the tissue depth of the lesion to be imaged, and the type of instrumentation employed.
  • FDPM frequency-domain photon migration
  • the FDPM method is as follows:
  • step (d) generating an image of the tissue by mapping the heterogeneous composition of the tissue in accordance with the values of step (c).
  • the fluorescence characteristic of step (c) preferably corresponds to uptake of the imaging agent and preferably further comprises mapping a number of quantities corresponding to adsorption and scattering coefficients of the tissue before administration of the agent.
  • the fluorescence characteristic of step (c) preferably corresponds to at least one of fluorescence lifetime, fluorescence quantum efficiency, fluorescence yield and imaging agent uptake.
  • the fluorescence characteristic is preferably independent of the intensity of the emission and independent of imaging agent concentration.
  • the quantifying of step (c) preferably comprises: (i) establishing an estimate of the values, (ii) determining a calculated emission as a function of the estimate, (iii) comparing the calculated emission to the emission of said detecting to determine an error, (iv) providing a modified estimate of the fluorescence characteristic as a function of the error.
  • the quantifying preferably comprises determining the values from a mathematical relationship modelling multiple light-scattering behaviour of the tissue.
  • the method of the first option preferably further comprises monitoring a metabolic property of the tissue in vivo by detecting variation of said fluorescence characteristic.
  • the optical imaging of the sixth aspect is preferably used to help facilitate the management of a disease state of the mammalian body.
  • management is meant use in the: detection, staging, diagnosis, monitoring of disease progression or the monitoring of treatment.
  • the disease state is suitably one in which the BTM of the imaging agent is implicated.
  • Disease states are preferably located near the body surface or in body cavities or can be exposed by surgical procedures.
  • the present invention provides a method of detection, staging, diagnosis, monitoring of disease progression or monitoring of treatment of a disease state of the mammalian body which comprises the in vivo optical imaging method of the sixth aspect.
  • the present invention provides a method for the assay of an analyte in a sample which method comprises:
  • the assay method is a direct assay for the measurement of an analyte in a sample.
  • a known or putative inhibitor compound may be included in the assay mix, in which case, the measurement may be correlated with the biological activity of the known or putative inhibitor.
  • the assay may be a competitive assay, wherein a sample containing an analyte competes with the fluorescent conjugate for a limited number of binding sites on a binding partner that is capable of specifically binding both the analyte and the conjugate. Increasing amounts (or concentrations) of the analyte in the sample will reduce the amount of the fluorescent conjugate of Formula II that is bound to the specific binding partner. The fluorescence signal is measured and the concentration of analyte may be obtained by interpolation from a standard curve.
  • the binding assay may employ a two-step format, wherein a first component, which may be optionally coupled to an insoluble support, is bound to a second component to form a specific binding complex, which is bound in turn to a third component.
  • the third component is capable of specifically binding to either the second component, or to the specific binding complex.
  • Either of the second or the third component may be the conjugate of the present invention. Examples include "sandwich" assays, in which one component of a specific binding pair, such as a first antibody, is coated onto a surface, such as the wells of a multiwell plate.
  • a fluorescent labelled second antibody is added to the assay mix, so as to bind with the antigen- first antibody complex.
  • the fluorescence signal is measured and the concentration of antigen may be obtained by interpolation from a standard curve.
  • analyte-specific binding partner pairs include, but are not restricted to, antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that any molecules which possess a specific binding affinity for each other may be employed, so that the fluorescent dyes of the present invention may be used for labelling one component of a specific binding pair, which in turn may be used in the detection of binding to the other component.
  • the compounds of the present invention may also be used in a detection method wherein a plurality of the fluorescent dyes are covalently attached to a plurality of different primary components, such as antibodies, each primary component being specific for a different secondary component, such as an antigen, in order to identify each of a plurality of secondary components in a mixture of secondary components.
  • each of the primary components is separately labelled with a fluorescent dye having a different light absorption and emission wavelength characteristic, compared with the dye molecules used for labelling the other primary components.
  • the labelled primary components are then added to the preparation containing secondary components, such as antigens, and the primary components are allowed to attach to the respective secondary components for which they are selective.
  • any unreacted probe materials may be removed from the preparation by, for example, washing, to prevent interference with the analysis.
  • the preparation is then subjected to a range of excitation wavelengths including the absorption wavelengths of particular fluorescent compounds.
  • a fluorescence microscope or other fluorescence detection system such as a flow cytometer or fluorescence spectrophotometer, having filters or monochromators to select the rays of the excitation wavelength and to select the wavelengths of fluorescence is next employed to determined the intensity of the emission wavelengths corresponding to the fluorescent compounds utilized, the intensity of fluorescence indicating the quantity of the secondary component which has been bound with a particular labelled primary component.
  • Known techniques for conducting multi-parameter fluorescence studies include, for example, multiparameter flow cytometry.
  • a single wavelength of excitation can be used to excite fluorescence from two or more materials in a mixture where each fluoresces at a different wavelength and the quantity of each labelled species can be measured by detecting its individual fluorescence intensity at its respective emission wavelength.
  • a light absorption method can also be employed.
  • the detection method of the present invention can be applied to any system in which the creation of a fluorescent primary component is possible.
  • an appropriately reactive fluorescent compound can be conjugated to a DNA or RNA fragment and the resultant conjugate then caused to bind to a complementary target strand of DNA or RNA.
  • Appropriate fluorescence detection equipment can then be employed to detect the presence of bound fluorescent conjugates.
  • Examples 1 to 7 provide the syntheses of various fluorinated dye precursors of Formula A of the present invention, which can be reacted with components (Ha) and (C) as described above to give dyes of the invention.
  • Examples 8 and 9 provide [benzo[b]pyrilium compounds of Formula (Ha), suitable for coupling with the precursors of Examples 1 to 7.
  • Example 10 provides a method of N-alkylation of heterocyclic N atoms to introduce 4-sulfobutyl substituents.
  • Examples 11 to 23 provide dyes of Formulae III, IVb and IVc of the invention.
  • Examples 11-18 provide the syntheses of pentamethine dyes of the invention.
  • Examples 19-21 provide the syntheses of trimethine dyes of the invention.
  • Examples 22-23 demonstrate the synthesis of heptamethine dyes of the invention.
  • Examples 24 and 25 provide dyes of the invention substituted with an NHS ester (Q a group).
  • Example 26 provides a photostability study demonstrating that the fluorinated dyes generally demonstrate greater resistance to photobleaching when compared with the non-fluorinated dye analogues. Control samples that were not exposed to light over the same time period, showed no reduction in absorbance.
  • Example 27 provides the synthesis of RGD peptide conjugates of dyes of the invention. Figures 1 and 2 show the improved photostability of dyes of the invention.
  • DMF Dimethylformamide HATU: O-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • Example 1 4-f l-(5-carboxypenM)-4,5,6,7-tetrafluoro-2,3-dimethyI-3H- indolium-3-yllbutane-l-s ⁇ lfonate.
  • 2,3,4,5-Tetrafluoroaniline (1.75g, 0.01M) was dissolved in cone. HCl (280ml).
  • the flask was maintained at -10 0 C and a solution OfNaNO 2 (leq) in water (10ml) added dropwise followed subsequently by a solution of tin(II) chloride (3.4g) in cone. HCl (40ml).
  • the reaction was returned to ambient temperature and stirred for 1 hr.
  • the solvent was removed in vacuo to yield crude 2,3,4,5-tetrafluorophenylhydrazine hydrochloride as a yellow solid (7g).
  • Example 2 4-f3-(5-carboxypentyl)-4,5,6,7-tetrafluoro-2,3-dimet]ryl-3H- mdolium-l-yllbiitane-l-sulfonate.
  • Example 4 4-f3-(5-carboxypentyI)-2,3-dimethyl-4,6-6/5(trifluoromethyl)-3jH r - mdoIium-1 -yll butane-1 -sulf on ate.
  • Example 5 4- ⁇ 3-(5-carboxypentyl)-6-flttoro-2,3-dimethyl-4-(trifluoromethyl)- 3H-indolium-l-vIlbutane-l-sttlfonate
  • Example 6 4-fl-(5-carboxypentvI)-6-fluoro-2,3-dimethvI-4-(trifluoromethyl)- 3ff-indolium-3-yIlbiitane-l-siiIfonate.
  • Example 7 4-[l-(5-carboxypentyl)-5,7-diflttoro-2.,3-dimethyI-3H r -indolium-3- ylibutane-l-sulfonate.
  • Example 8 2-fer ⁇ ButyI-7-dimethvIamino-4-methylchromenyIium tetrafluoroborate.
  • Example 9 1 l-fe ⁇ butyI-9-methyI-23,6 J-tetrahvdro4H n 5H ⁇ -pyrano ⁇ 2 ⁇ ,3- /1pyridof3,24-// ⁇ ⁇ quinolm-124I ⁇ m tetrafl ⁇ oroborate.
  • Example 10 General method for N-alkylation of quinaldine, Iepidine and acridine with butane sultone.
  • N-Alkylation of quinaldines, lepidines and 9-methyl-acri dines with butane sultone to form N-(4-sulfonatobutyl)-derivatives may be performed by a method analogous to that described in US Patent No. 6579718 (Yue et at).
  • a mixture of the nitrogen heteroaromatic and butane sultone (excess) are heated at up to 14O 0 C for up to 24 hours whilst stirring. After cooling to room temperature the mixture is triturated with ethyl acetate or diethyl ether to yield the N-alkylated product, which is used directly in dye synthesis.
  • the following example describes a typical synthesis.
  • Example 11 4-f(4F)-4-((2J?,4£l-5-ri-(5-carboxypentyl)-4.5,6J-tetranuoro-3- methyI-3-(4-sttlfobutyI)-3i?-indoImm-2-vnpenta-2,4-dienvIidene ⁇ quinolm-l(4jy)- yli butane-1 -sulf on ate.
  • Example 12 4-r(2F)-2-U2E,4F)-5-fl-(5-carboxypentvn-4,5,6J-tetrafluoro-3- methyl-3-(4-sulfobutyI)-3jH-indolimn-2-vnpenta-2,4-dienylidene
  • Example 13 4-f(2J5')-2- ⁇ f2£',4 ⁇ -5-ri-(5-carboxypeiitvI)-4,5,6.,7-tetrafIuoro-3- methyl-3-(4-suIfobutvI)-3H r -indoIium-2-vIlpenta-2,4-dienyIidene ⁇ -6- methyIqumoIm-l(2H)-yIlbutane-l-sttlfonate.
  • Example 14 4-r(2 ⁇ )-2- ⁇ r2E,4E)-5-fl-f5-carboxypentvI)-4,5,6,7-tetranuoro-3- methyl-3-(4-sulfobutyI)-3iJ-indoIium-2-vnpenta-2,4-dienyIidene ⁇ -6- fluoroqumoIin-l(2H)-yI1butane-l-sulfbnate.
  • Example 15 ⁇ rflFl-l-ffl ⁇ -S-ri-fS-carboxypentvIM ⁇ J-tetrafluoro-B- methyl-3-(4-sulfobutyl)-3H r -indolimn-2-vIlpenta-2,4-dienyIidene ⁇ -7- fluoroqumolm-l(2H)-vnbutane-l-sulfonate.
  • Example 16 4-r(2 J g)-2- ⁇ (2E,4E)-5-[l-(5-carboxypentvD-4,5.,6J-tetrafluoro-3- methyl-3-(4-suIfobutyIV3jff4ndolium-2-yl1penta-2,4-dienyIidene ⁇ -7- chioroquinolin-l(2ff)-vnbutane-l-sulfonate.
  • Example 17 442- ⁇ Q£3£.£ffi-542-fert-butyl-7-(dimethylammo)-4g-chromen- 4-yIidene1penta-l,3-dienvU-l-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyI- 3H-indolmm-3-yllbutane-l-sulfonate (Compound 1).
  • Example 18 4- ⁇ 2- ⁇ aE,3E,5E)-5-(ll-tert-butyl-2,3,6J-tetrahydro-lH ⁇ 5H,9H- pyrano[2,3-flpyridof3,2,l-//]quinoIin-9-ylidene)peiita-l,3-dienyn-l-(5- carboxypentvI)-4,5,6,7-tetrafluoro-3-niethyl-3H-indolimn-3-yllbutane-l- sulfonate.
  • Example 19 4-F2- ⁇ (lJ5',3£)-3-r2-te/'f-butvI-7-(dimethylamino)-4H-chromeii-4- ylidene1prop-l-e ⁇ yll-l-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3/f- indoIium-3-vnbutane-l-sulfonate (Compound 3).
  • Example 20 4-[2- ⁇ (l£',3-g)-3-[2-fe ⁇ -butyl-7-(dimethylaminoV4H r -chromen-4- ylideneiprop-l-enyll-l-CS-carboxypentvD-S ⁇ -difluoro-B-methyl-S. ⁇ -indolium-S- yllbiitane-l -sulfonate.
  • Example 21 4- ⁇ 2- ⁇ (lE,3E)-3-(ll-tert-bntyl-2,3,6J-tetrah ⁇ dro-lH,5H,9H- pyrano[2.,3-/lPVridof3.,2,l-t/1quinoliii-9-ylidene)prop-l-enyn-l-(5- carboxypentyl)-5.,7-difluoro-3-methyl-3H-indoliuin-3-vnbutane-l-sttIfonate.
  • Example 22 4-r(2E)-2-((2 ⁇ ,4E,6E)-7-ri-(5-carboxypentvn-5,7-difluoro-3- methyl-3-(4-sulfobutyl)-3/r-indoIium-2-yllhepta-2,4,6-trienyUdene ⁇ -6- methylquinolin-l(2iT)-yIlbutane4 -sulfonate.
  • Example 23 4-[2- ⁇ (ljg.,3E,5£'JE)-7-[2-f ⁇ yf-butyl-7-fdimethvIamino)-4H- chromen ⁇ -ylideneihepta-l ⁇ .S-trienyll-l-fS-carboxypenty ⁇ -SJ-difluoro-S- methvI-3H-indoIimn-3-vnbutane-l-suIfonate.
  • Example 24 4-(2-j(lJ5',3£',5jg)-5-r2-te ⁇ -butyl-7-(dimethylaminoV4H-chromeii-4- yIidenelpenta-l,3-dienvU-l- ⁇ 6-[(2. ⁇ 5-dioxopyrroIidin-l-yl)oxy1-6-oxohexyI ⁇ - 4,5,6,7-tetrafluoro-3-methyl-3i?-indolimn-3-yl)butane-l-suIfonate.
  • Example 25 4-(2- ⁇ (lEJE,5E)-5-(l l-fert-butyI-2,3,6 J-tetrahydro-lH.Sg ⁇ H- pyranof2.,3-/1PVrido[3,2,l-//lquinolin-9-vIidene)peiita-l,3-dienvIl-l- ⁇ 6-f(2,5- dioxopyrroIidin-l-yl)oxyl-6-oxohexyU-4,5,6,7-tetrafluoro-3-methyI-3ff- indolium-3-yDbiitane-l-sttlfbnate.
  • Example 26 Photostat) ilitv of Dyes of the Invention. Photostability studies were performed as detailed below. Each test dye was dissolved in either a 1 :1 mixture of methanol: water or water to give an absorbance reading between 0.7 and 1.2Au. Each solution was then divided into 2 further vials. One vial of dye solution was maintained in a dark environment as a control sample ("dark controls") during the course of the experiment. The other was exposed to a strong light source (a Wallac light box; 1295-013). Samples were maintained at 22cm above the light source, with continuous exposure to light. The UV/Visible spectrum of each sample was measured at regular intervals over a six day trial period. The same cuvettes and spectrophotometer were used for each measurement point. For the control samples maintained in the dark, the UV/visible absorption spectra were measured at both the start and at the end of the experiment. Each test was performed in duplicate.
  • a strong light source a Wallac light box
  • the dyes were dissolved in a 1 :1 mixture of methanol: water to give an absorbance reading of IAu at the dyes lambda max and then diluted 20 times for fluorescence measurements.
  • the samples were exposed to light with a Karl Storz Xenon 175 light source at 100% intensity for 30 min.
  • the cuvette was kept in a water bath while exposed to light, to avoid heating. Fluorescence measurements were made on Cary Eclipse (Varian) fluorescence spectrophotometer 1 cm cuvette, ex/em slit 5 nm
  • the peptide shown was assembled using standard solid phase peptide synthesis methods.
  • the chloroacetylated peptide was cleaved from the solid support and cyclised in solution, first forming the thioether bridge and then the disulphide bridge.
  • Compound 1 (Example 17; 0.0018 mmol) was dissolved in NMP (0.5 mL) and NMM (1 ⁇ L) was added followed by HATU (0.84 mg, 0.0022 mmol). The solution was stirred in the dark for 5 minutes and then added to a solution of RGD peptide amine (2.3 mg, 0.0018 mmol) in NMP (0.5 mL). The reaction mixture was stirred for five hours at room temperature then heated to 50 0 C for two hours.
  • Conjugate 2 was prepared in a similar manner using the dye of Example 18.
  • conjugate 1 the reaction mixture was then diluted with 5 % MeCN/water (5 mL) and the product purified using preparative HPLC.

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